title: Retinitis pigmentosa mutants provide insight into the role of the N-terminal cap in rhodopsin folding, structure, and function.
abstract: Autosomal dominant retinitis pigmentosa (ADRP) mutants (T4K, N15S, T17M, V20G, P23A/H/L, and Q28H) in the N-terminal cap of rhodopsin misfold when expressed in mammalian cells. To gain insight into the causes of misfolding and to define the contributions of specific residues to receptor stability and function, we evaluated the responses of these mutants to 11-cis-retinal pharmacological chaperone rescue or disulfide bond-mediated repair. Pharmacological rescue restored folding in all mutants, but the purified mutant pigments in all cases were thermo-unstable and exhibited abnormal photobleaching, metarhodopsin II decay, and G protein activation. As a complementary approach, we superimposed this panel of ADRP mutants onto a rhodopsin background containing a juxtaposed cysteine pair (N2C/D282C) that forms a disulfide bond. This approach restored folding in T4K, N15S, V20G, P23A, and Q28H but not T17M, P23H, or P23L. ADRP mutant pigments obtained by disulfide bond repair exhibited enhanced stability, and some also displayed markedly improved photobleaching and signal transduction properties. Our major conclusion is that the N-terminal cap stabilizes opsin during biosynthesis and contributes to the dark-state stability of rhodopsin. Comparison of these two restorative approaches revealed that the correct position of the cap relative to the extracellular loops is also required for optimal photochemistry and efficient G protein activation.
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Essex CO4 3SQ, United Kingdom.
authors.forename: Chikwado A
authors.initials: CA
authors.lastname: Opefi
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Essex CO4 3SQ, United Kingdom.
authors.forename: Kieron
authors.initials: K
authors.lastname: South
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Essex CO4 3SQ, United Kingdom.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Reynolds
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215.
authors.forename: Steven O
authors.initials: SO
authors.lastname: Smith
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Essex CO4 3SQ, United Kingdom. Electronic address: preeves@essex.ac.uk.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Reeves
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012172:Retinaldehyde / Q000235:genetics / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000235:genetics / Q000378:metabolism*; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism*
chemical_list: C013110:metarhodopsins; D012243:Rhodopsin; D012172:Retinaldehyde
keywords: G-protein-coupled Receptor (GPCR); Membrane Proteins; Phototransduction; Protein Misfolding; Protein Stability; Retinal Degeneration; Retinoid; Rhodopsin